Department of Neurodegenerative Diseases, Hertie-Institute for Clinical Brain Research, University of Tübingen, Germany German Research Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Germany matthis.synofzik@uni-tuebingen.de.

Department of Neurodegenerative Diseases, Hertie-Institute for Clinical Brain Research, University of Tübingen, Germany German Research Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Germany.

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Department of Neurodegenerative Diseases, Hertie-Institute for Clinical Brain Research, University of Tübingen, Germany German Research Center for Neurodegenerative Diseases (DZNE), University of Tübingen, Germany Dr. John T. Macdonald Foundation Department of Human Genetics and John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, USA.

Abstract

Mutations in the synaptic nuclear envelope protein 1 (SYNE1) gene have been reported to cause a relatively pure, slowly progressive cerebellar recessive ataxia mostly identified in Quebec, Canada. Combining next-generation sequencing techniques and deep-phenotyping (clinics, magnetic resonance imaging, positron emission tomography, muscle histology), we here established the frequency, phenotypic spectrum and genetic spectrum of SYNE1 in a screening of 434 non-Canadian index patients from seven centres across Europe. Patients were screened by whole-exome sequencing or targeted panel sequencing, yielding 23 unrelated families with recessive truncating SYNE1 mutations (23/434 = 5.3%). In these families, 35 different mutations were identified, 34 of them not previously linked to human disease. While only 5/26 patients (19%) showed the classical SYNE1 phenotype of mildly progressive pure cerebellar ataxia, 21/26 (81%) exhibited additional complicating features, including motor neuron features in 15/26 (58%). In three patients, respiratory dysfunction was part of an early-onset multisystemic neuromuscular phenotype with mental retardation, leading to premature death at age 36 years in one of them. Positron emission tomography imaging confirmed hypometabolism in extra-cerebellar regions such as the brainstem. Muscle biopsy reliably showed severely reduced or absent SYNE1 staining, indicating its potential use as a non-genetic indicator for underlying SYNE1 mutations. Our findings, which present the largest systematic series of SYNE1 patients and mutations outside Canada, revise the view that SYNE1 ataxia causes mainly a relatively pure cerebellar recessive ataxia and that it is largely limited to Quebec. Instead, complex phenotypes with a wide range of extra-cerebellar neurological and non-neurological dysfunctions are frequent, including in particular motor neuron and brainstem dysfunction. The disease course in this multisystemic neurodegenerative disease can be fatal, including premature death due to respiratory dysfunction. With a relative frequency of ∼5%, SYNE1 is one of the more common recessive ataxias worldwide.

SYNE1 mutations . ( A ) Graphical overview of the mutations found in this study in relation to the SYNE1 domains. Numbers indicate the mutation IDs of the mutations identified in this study ( ). Their position indicates the position of the respective mutations in the SYNE1 gene. Blue = N-terminal actin binding domain [calponin homology domains containing actin binding sites (IPR001715)] and mutations affecting this domain; orange = spectrin/alpha-actinin repeat domains (IPR018159) and mutations affecting these domains; turquoise = KASH domain (C-terminal klarsicht domain) (IPR012315); black = mutations not affecting any of these domains. Mutation 33 (black circle) is the only missense mutation in the present study. ( B ) Overview of the variant types and their location of all published and novel SYNE1 mutations. The presentation of the giant SYNE1 gene is split in a first part (chr6:152.442.819–152.644.000; top ) and a second part (chr6:152.644.000–152.958.534; bottom ). It presents the variant types of all SYNE1 mutations found in the present study ( bottom row of each panel) and other studies (Human Gene Mutation Database) ( top row of each panel), their location and their annotation with the associated clinical phenotypes. ATX = ataxia; AMC = arthrogryposis multiplex congenita; ED = Emery-Dreifuss muscular dystrophy; CM = cardiomyopathy; MR = mental retardation; HSP = hereditary spastic paraplegia. Note that, except for ATX and AMC, all other phenotypes have been associated only with missense mutations, not truncating mutations in SYNE1 . Green coloured arrows and boxes = indel mutations; blue coloured arrows and boxes = stop mutations; red coloured arrows and boxes = splice site mutations; purple coloured arrow and box = missense mutation. HG19 genome build. Transcript: NM_033071 > NP_149062.

Severely reduced to absent SYNE1 staining in muscle tissue of SYNE1 patients . Immunohistological findings in control tissue ( A , E and I ), as well as in the quadriceps muscle biopsies of three different SYNE1 patients from three different countries: Patient 13-1, Belgian ( B , F and J ); Patient 10-1, German ( C , G and K ); and Patient 6-1, Italian ( D , H and L ). All three patients show a severely reduced to absent staining of the nuclear envelope after immunolabelling of nesprin-1 ( B – D ), whereas staining was normal in the control ( A ) (peroxidase-antiperoxidase technique). To control for an unspecific lack of staining in the SYNE1 patients, further control stainings were performed. Immunolabelling of emerin and lamin A/C at the inner nuclear membrane was normal in all three patients as well as the control ( E – H and I – L , respectively) (avidin-biotin complex technique).

The phenotypic spectrum of SYNE1 disease. ( A ) Frequency of non-cerebellar features in SYNE1. Relative frequencies of non-cerebellar features observed in the n = 26 SYNE1 patients investigated in this study. Note that several patients showed ≥2 non-cerebellar disease features. ( B ) The relative share of main SYNE1 phenotypes. This figure illustrates that only a minor share of SYNE1 patients shows pure cerebellar ataxia (blue), whereas the large majority show cerebellar ataxia plus phenotypes (yellow), in particular cerebellar ataxia plus motor neuron disease (MND). The latter combination is thereby not a distinct ataxia plus-phenotype, but is commonly associated also with other non-motor neuron disease signs (see overlap group comprising of n = 10 subjects). ( C ) The continuous spectrum of SYNE1 disease. The clinical spectrum of neurological and non-neurological SYNE1 features presented in A unfolds along a continuous spectrum of disease. Variable combinations of these features can be found in different SYNE1 patients reaching from pure cerebellar ataxia ( left ) via cerebellar ataxia plus damage of one or two additional systems [e.g. motor neuron disease (MND)] to cerebellar ataxia as part of severe multisystemic neurodegenerative syndrome ( right ). In addition to ataxia and motor neuron disease, the latter can include also mental retardation, respiratory distress and visceral organs. The phenotype of mild, slowly progressive pure cerebellar ataxia, which has been considered the most prominent phenotype of SYNE1 so far ( ; ), thus represents only one far edge of this broad disease spectrum. CK = creatine kinase.

Cerebral 18 F- FDG PET and MRI in exemplary SYNE1 patients. ( A and C ) Surface projections of the 18 F-FDG PET scans of Patient 10-1 at age 37 years ( A ) and Patient 9-1 at age 45 years ( C ). The upper rows in A and C show the surface projection map of cerebral glucose metabolism normalized to the maximum of the acquisition. The lower rows show the deviation of an age-adjusted normal database after normalization to a global mean calculation (GLB) of metabolic activity. On both projections, a marked homogenous bilateral reduction of cerebellar FDG metabolism can be seen in both subjects. ( B and D ) Tomographic projections after automated fitting. For both Patient 10-1 ( B ) and Patient 9-1 ( D ) a reduction of FDG-metabolism is also clearly visible in the brainstem (white arrows), which reached statistical significance in Patient 10 with a Z-score of 3 and a trend in Patient 9-1 with a Z-score of 1.5 (semiquantitative PET, deviation after normalization to the global mean). ( E and F ) T 2 -weighted MRI scans in Patient 18-1 at age 21 years shows marked vermian atrophy ( F ) and cortical hemisphere atrophy ( E ) of the cerebellum (red arrows). ( G ) Fusion image of 18 F-FDG PET scan of Patient 10-1 onto a central paramedian sagittal T 2 -weighted MRI scan shows considerable hypometabolism in the cerebellum and in the pons, whereas FDG metabolism is normal in the cortical cerebral regions.